JPH0236234B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field] The present invention relates to a method for stabilizing pullulanase. [Prior art] Pullulanase is an enzyme that cleaves the α-1,6-glucoside bond of pullulan and ultimately produces maltotriose. It is known that Pullulanase can also degrade α-1,6-glucoside bonds present in amylopectin, starch, and these microorganisms;
By acting on starch in combination with β-amylase and various α-1,6-amylases that do not have the ability to decompose 1,6-glucoside bonds, each amylase can be used in a higher yield than when these amylases are used alone. You can get products. for example,
When β-amylase acts alone on starch, the yield of maltose is about 55%, but when it acts in the presence of pullulanase, maltose can be produced with an extremely high yield of about 90%. Recently, it has been revealed that when pullulanase is used in combination with glucoamylase, glucose can be produced at a higher yield than when glucoamylase is used alone.
57−174089 and others). In this case, pullulanase is thought to act effectively to increase the yield of glucose by promoting the starch saccharification reaction by supplementing the α-1,6-glucoside bond cleaving ability of glucoamylase. However, glucoamylase differs slightly depending on its microbial origin, but generally has a pH of 4 to 4.
5. It is an enzyme that acts most efficiently in the temperature range of 55-60°C. On the other hand, many of the pullulanases known so far have a pH of 6 to 7, a temperature of
The optimal action range is between 50 and 55°C, which does not match the action range of glucoamylase. Therefore, the reaction had to be carried out at the expense of some enzyme activity. [Purpose and effect] The present inventors have conducted various studies on methods for stabilizing pullulanase to make it act in the preferred action range of glucoamylase. It was found that pullulanase was significantly stabilized when treated with dicarboxylic acid, particularly adipic acid (hexane dioxylic acid) having 6 carbon atoms. The present invention has been made based on this knowledge. [Structure] That is, the present invention relates to a method for stabilizing pullulanase, which is characterized by treating pullulanase with a dicarboxylic acid. The contents of the present invention will be explained in more detail below. When the present invention is applied to, for example, pullulanase used in combination with glucoamylase, the pullulanase is required to act at least at an optimum temperature of 50° C. or higher (1% pullulan concentration) and at a pH of 5. Such enzymes are produced by various microorganisms such as Bacillus, Klebsilla (Aerobacter), and Streptomyces; for example, Klebsilla pneumonia.
Bacillus subtilis pullulanase and Bacillus subtilis pullulanase are used. Examples of dicarboxylic acids used in the present invention include adipic acid (6 carbon atoms), pimelic acid (7 carbon atoms), suberic acid (8 carbon atoms), azelaic acid (9 carbon atoms), and zebacic acid (10 carbon atoms). It is a saturated dicarboxylic acid represented by the general formula CnH ₂ n (CooH) ₂ such as 5 to 10 carbon atoms, and adipic acid is particularly effective. When treating pullulanase with adipic acid, the pullulanase agent is usually treated in advance by adding dicarboxylic acid in an amount of 0.5 to 5 mmol per unit of the enzyme, or it is added to the enzyme reaction solution. The pullulanase agent treated in this way acts effectively at pH 4.3-4.5 and temperature 57-60°C, which are the preferred action conditions for glucoamylase, especially Aspergillus niger glucoamylase, and is non-active. Glucose yield can be increased by 0.5 to 3% compared to treatment with pullulanase. Next, the details of the present invention will be explained with reference to Examples. Example 1 Soluble starch 2%, urea 0.35%, K ₂ HPO ₄ 0.05%
_MgSO4・_7H2O0.05 %, KCl0.5%, _MnCl2 5×
200 ml of 50 ml of medium consisting of 10 ^-5 M, Cal ₂ l x 10 ^-3 M
Pour into an Erlenmeyer flask, sterilize using a conventional method, and remove Klebsiella pneumonia.
pneumoniae) FERMP-7387 was inoculated and cultured with shaking at 30°C for 2 days. After culturing, the pullulanase activity of the supernatant obtained by centrifugation was 6.0 per ml of medium.
It was a unit. 2 units of adipic acid per unit of pullulanase.
After adding millimolar amount and treating at room temperature for 30 minutes, it was used as a saccharification reaction enzyme. Commercially available dextrin (Powdered starch syrup SLD manufactured by Sanmatsu Kogyo Co., Ltd.) 3.8 g, glucoamylase (Amyloglucosidase AMG300 manufactured by Novo, Denmark)
Add 2.2 x 10 ^-3 ml, calcium chloride 1 x 10 ^-2 M, the above adipic acid treatment or untreated pullulanase at 0.75 units per g of starch, make the total volume 10 ml with water, pH 4.5, temperature 60℃. The reaction was carried out. The yield of glucose 55 hours after the start of the reaction was analyzed by high performance liquid chromatography, and the results are shown in Table 1.

[Table] As is clear from the table, pullulanase treated with adipic acid had a remarkable effect on increasing glucose yield compared to untreated pullulanase. Example 2 Soybean meal 5%, corn, stave liquor 0.6
% meat extract 0.4%, dipotassium phosphate 0.3%, magnesium sulfate (heptahydrate) 0.1%, soluble starch 2%, urea 0.3%, sodium dodecyl sulfate
0.1%, copper sulfate 5×10 ^-5 , zinc sulfate 1×10 ^-4 M,
200ml of 30ml medium (PH7.2) consisting of iron sulfate 1×10 ^-5
Pour into a ml Erlenmeyer flask, sterilize it by a conventional method, and add Bacillus subtilit TU.
(FERM BP684) was inoculated and cultured at 30°C for 4 days. After culturing, the pullulanase activity in the supernatant obtained by centrifugation was 10.5 units per ml of medium. Adipic acid, pimelic acid, suberic acid, azelaic acid, and zebacic acid were added in an amount of 4 mmol each per unit of pullulanase, and the mixture was treated at room temperature for 30 minutes before being used for saccharification. The starch saccharification reaction was performed using the same composition as the method described in Example 1 except for using the above-mentioned pullulanase.
The reaction was carried out at 60°C. The yield of glucose measured 53 hours after the start of saccharification by high performance liquid chromatography was as shown in Table 2.

[Table] As is clear from the table, those treated with each dicarboxylic acid were found to be more effective than those not treated, and those treated with adipic acid showed particularly remarkable effects. Example 3 Adipic acid was added in amounts of 1, 2, 3, and 4 mmol to each unit of the enzyme solution prepared in Example 2, and after treatment at room temperature for 30 minutes, the composition was the same as in Example 1, pH 4.4. The reaction was carried out at 60°C for 2 days. The sugar composition of the obtained saccharified liquid was as shown in Table 3.

【table】

Claims (1)

[Claims] 1. A method for stabilizing pullulanase, which comprises treating pullulanase with a dicarboxylic acid.